This invention relates generally to the identification of a new superfamily of eukaryotic protein kinases and the use of one member of this superfamily, elongation factor-2 kinase (eEF-2 kinase), in assays to screen for specific inhibitors. Specific inhibitors of the eEF-2 kinase may be potent therapeutics for amelioration of malignant transformation. Additionally, sequences complementary to eEF-2 kinase may have therapeutic efficacy as antisense drugs or be used in gene therapy. Specifically, the invention relates to assays developed using the recombinant eEF-2 kinase to screen for inhibitors of phosphorylation of a peptide derived from the myosin heavy chain (MHC) protein.
Protein phosphorylation plays a critical role in many cellular processes (Krebs (1994) Trends Biochem. Sci. 19:439; Hanks and Hunter, (1996) FASEB J. 9:576-596; Hardie and Hanks, (1995) The Protein Kinase Facts Book (Academic, London)). There are two well-characterized superfamilies of protein kinases, with most of the protein kinases belonging to the serine/threonine/tyrosine kinase superfamily (Hanks and Hunter, (1996); Hardie and Hanks, (1995)). The characterization of several hundred members of this superfamily revealed that they all share a similar structural organization of their catalytic domains which consist of twelve conserved subdomains (Hanks and Hunter, (1996); Hardie and Hanks, (1995)). The other superfamily is referred to as the histidine kinase superfamily and is involved in the prokaryotic two-component signal transduction system, acting as sensor components (Stock et al., (1989) Microbiol. Rev. 53:450-490; Parkinson and Kofoid, (1992) Annu. Rev. Genet. 26:71-112; Swanson, et al., (1994) Trends Biochem. Sci. 19:485-490). Recently, eukaryotic members of this superfamily have also been described (Chang et al., (1993) Science 263:539-544; Ota and Varshavsky, (1993) Science 262:566-569; Maeda et al., (1994) Nature 369:242-245). Mitochondrial protein kinases have also recently been described that show structural homology to the histidine kinases, but phosphorylate their substrates on serine (Popov et al., (1992) J. Biol. Chem. 267:13127-13130; Popov et al., (1993) J. Biol. Chem. 268:26602-22606). Finally, several new protein kinases have been reported that show a lack of homology with either of the kinase superfamilies (Maru and Witte, (1991) Cell 67:459-468; Beeler et al., (1994) Mol. Cell. Biol. 14:982-988; Dikstein et al., (1996) Cell 84:781-790; Futey et al., (1995) J. Biol. Chem. 270:523-529; Eichenger et al., (1996) EMBO J. 15:5547-5556). However, these protein kinases are viewed as an exception to the general rule as they have yet to be fully characterized.
The cloning and sequencing of the extensively characterized eukaryotic elongation factor-2 kinase (eEF-2 kinase) from a variety of eukaryotic organisms has now revealed the existence of a novel class of protein kinases (Ryazanov et al., (1997) Proc. Natl. Acad. Sci., USA 94:4884-4889). eEF-2 kinase, previously known as Ca2+/calmodulin-dependent protein kinase III, is highly specific for phosphorylation of elongation factor-2 (eEF-2), an abundant cytoplasmic protein that catalyzes the movement of the ribosome along mRNA during translation in eukaryotic cells (reviewed in Ryazanov and Spirin, (1993) In Translational Regulation of Gene Expression (Plenum, New York) Vol. 2, pp. 433-455; Nairn and Palfrey, (1996) In Translational Control (CSHL Press, New York) pp. 295-318). All mammalian tissues, and various invertebrate organisms, exhibit eEF-2 kinase activity (Abdelmajid et al., (1993) Int. J. Dev. Biol. 37:279-290). eEF-2 kinase catalyzes the phosphorylation of eEF-2 at two highly conserved threonine residues located within a GTP-binding domain (Ryazanov and Spirin, (1993) In Translational Regulation of Gene Expression (Plenum, New York) Vol. 2, pp. 433-455; Nairn and Palfrey, (1996) In Translational Control (CSHL Press, New York) pp. 295-318). When eEF-2 is phosphorylated, it becomes inactive with respect to protein synthesis (Ryazanov et al., (1988) Nature 334:170-173). Since eEF-2 phosphorylation is dependent on Ca2+ and calmodulin, eEF-2 kinase plays a pivotal role in modulating the protein synthesis rate in response to changes in intracellular calcium concentration. Phosphorylation of eEF-2 has also been linked to the regulation of cell cycle progression. For example, transient phosphorylation of eEF-2 occurs during the mitogenic stimulation of quiescent cells (Palfrey et al., (1987) J. Biol. Chem. 262:9785-9792) and during mitosis (Celis et al., (1990) Proc. Natl. Acad. Sci., USA 87:4231-4235). In addition, changes in the level of eEF-2 kinase activity is associated with a host of cellular processes such as cellular differentiation (End et al., (1982) J. Biol. Chem. 257:9223-9225; Koizumi et al., (1989) FEBS Lett. 253:55-58; Brady et al., (1990) J. Neurochem. 54:1034-1039), oogenesis (Severinov et al., (1990) New Biol. 2: 887-893), and malignant transformation (Bagaglio et al., (1993) Cancer Res. 53:2260-2264).
The sequence eEP-2 kinase appears to have no homology to either the Ca2+/calmodulin-dependent protein kinases or to any members of the known protein kinase superfamilies (Ryazanov et al., (1997) Proc. Natl. Acad. Sci., USA 94:4884-4889). However, the recently described myosin heavy chain kinase A (MHCK A) from Dictyostelium (Futey et al., (1995) J. Biol. Chem. 270:523-529) shows a great deal of homology with eEF-2 kinase. These two kinases define a novel class of protein kinases that may represent a new superfamily.
Evidence for MHCK and eEF-2 kinase forming the core of a new superfamily is as follows. MHCK A from Dictyostelium, has a demonstrated role in the regulation of myosin assembly (Futey et al., (1995) J. Biol. Chem. 270:523-529; Cxc3x4txc3xa9 et al., (1997) J. Biol. Chem. 272:6846-6849). eEF-2 kinase is a ubiquitous Ca2+/calmodulin-dependant protein kinase involved in the regulation of protein synthesis by Ca2+ (Redpath et al., (1996) J. Biol. Chem 271:17547-17554; Ryazanov et al., (1997) Proc. Natl. Acad. Sci., USA 94:4884-4889). Both MHCK A and eEF-2 kinase display no homology to any of the known protein kinases, but are strikingly similar to each other; amino acid sequences of their catalytic domains are 40% identical. Another protein kinase homologous to MHCK A and eEF-2 kinase has recently been identified in Dictyostelium (Clancy et al., (1997) J. Biol. Chem. 272:11812-11815), and an expressed sequence tag (EST) sequence, with a high degree of similarity to the catalytic domain common to both MHCK A and eEF-2 kinase, has been deposited in GenBank (clone FC-AN09/accession #C22986). An amino acid sequence alignment of the catalytic domains of these new protein kinases is shown in FIG. 1A. These kinases have a catalytic domain of approximately 200 amino acids which can be subdivided into seven conserved subdomains. Subdomains V, VI, and VII have a predicted xcex2-sheet structure and are presumably involved in ATP-binding, while subdomains I through IV may be involved in substrate binding and catalysis. These new protein kinases have no homology to the members of the eukaryotic serine/threonine/tyrosine protein kinase superfamily with the exception of the GXGXXG motif in subdomain VI which is present in many ATP-binding proteins. Thus, MHCK A, eEF-2 kinase, and related protein kinases may represent a new superfamily. Evolutionary analysis of these new kinases (FIG. 1B) reveals that they can be subdivided into 2 families: the eEF-2 kinase family which includes eEF-2 kinases from different organisms, and the MHCK family which includes MHCK A, MHCK B and FC-AN09. These two families appear to have split more than a billion years ago.
An interesting question is why does nature employ these unusual kinases to phosphorylate eEF-2 and myosin heavy chains? Perhaps the answer is related to the secondary structure of the phosphorylation sites. As was originally reported by Small et al. (Small et al., (1977), Biochim. Biophys. Res. Comm. 79:341-346), phosphorylation sites are usually located at predicted xcex2-turns. Subsequent studies, including X-ray crystallographic data, demonstrated that phosphoacceptor sites in substrates of conventional protein kinases are often located in turns or loops and usually have flexible extended conformation (Knighton et al., (1991) Science 253:414-420; Pinna and Ruzzene (1996) Biochim. Biophys. Acta 1314:191-225). In contrast to this, the existing evidence suggests that the peptides around phosphorylation sites for eEF-2 kinases and MHCK A have an xcex1-helical conformation. The two major phosphorylation sites for MHCK A are located in a region which has a coiled-coil xcex1-helical structure (Vaillancourt et al., (1988) J. Biol. Chem. 253:10082-10087). The major phosphorylation site in eEF-2, threonine 56, is located within a sequence which is homologous among all translational elongation factors. In the crystal structure of the prokaryotic elongation factor EF-Tu, this sequence has an xcex1-helical conformation (Polekhina et al., (1996) Structure 4:1141-1151; Abel et al., (1996) Structure 4:1153-1159). These facts suggest that eEF-2 kinase and MHCK A differ from conventional protein kinases in that they phosphorylate amino acids located within xcex1-helices.
Thus, in addition to the two well-characterized superfamily of eukaryotic protein kinases, which phosphorylate amino acids located in loops and turns, there appears to be a third superfamily of xcex1-helix-directed kinases.
Novel protein kinase inhibitors have the potential to form the basis for pharmaceutical compositions that can ameliorate malignant transformation. In order to find these inhibitors, libraries of chemical compounds are routinely screened using an automated protein kinase assay. The drawback to this approach is that most protein kinases have a very similar structure, thus making it difficult to specific inhibitors which act solely on a particular protein kinase. We have recently determined the primary structure of eEF-2 kinase, a ubiquitous enzyme which is involved in the regulation of protein synthesis and the cell cycle. Unexpectedly, we found that eEF-2 kinase has a unique structure. It has no homology to any other mammalian protein kinase. This feature makes eEF-2 kinase an ideal target in the search for a specific protein kinase inhibitor. Since preliminary evidence suggests that eEF-2 kinase is upregulated in human cancers (data not shown), including, but not limited to, breast cancer, identification of specific inhibitors of eEF-2 kinase can eventually lead to the development of novel anticancer drugs. In order be able to perform a high throughput screen for an eEF-2 kinase inhibitor, it is first necessary to develop a simple assay which is amenable to automation. The existing assay involves incubation of partially purified eEF-2 kinase along with purified eEF-2 and [xcex3-32P]ATP as substrates in the presence of increasing concentrations of candidate inhibitors. Results are then obtained by electrophoretic separation of the reaction mixtures, followed by autoradiography. Results are then quantified by either densitometry or scintillation counting of excised bands from the gel containing 32P-eEF-2. Clearly, this assay, as it stands, is time-consuming, expensive, and not amenable to automation. Furthermore, it is difficult to purify large amounts of native eEF-2 required to perform multiple assays, and attempts to overexpress a recombinant form of eEF-2 were unsuccessful as its overexpression was toxic to host strains (personal communication from James Bodley, University of Minnesota, Minn.). Therefore, we have developed new methodologies for determining eEF-2 kinase activity, which involves the use of a specific peptide substrate; easily and economically manufactured in large scale. These methods are relatively inexpensive, fast, and can be fully automated.
In our first attempt to use a peptide as an eEF-2 kinase substrate, we generated peptides centered around the phosphorylation site of eEF-2. This strategy did not yield a peptide that was functional in phosphorylation assays (data not shown). Surprisingly, we found that a 16xe2x80x2 mer peptide (RKKFGESEKTKTKEFL (SEQ ID NO: 20)), based on the phosphorylation site of Dictyostelium discoideum MHC, was an acceptable substrate for use with eEF-2 kinase in phosphorylation assays. It is interesting to note that while eEF-2 kinase can phosphorylate a peptide derived from MHC, it is not able to phosphorylate native MHC (Ryazanov et al., (1997) Proc. Natl. Acad. Sci., USA 94:4884-4889).
In accordance with the present invention, a new superfamily of protein kinases and corresponding methods for assaying their phosphorylation activity are disclosed. The protein kinases of this new superfamily have the following characteristics: 1) No significant sequence homology to protein kinases of either the serine/threonine/tyrosine kinase or histidine kinase super families; 2) moderate to high (xe2x89xa740%) to eEF-2 kinases from any organism; and, 3) phosphorylates an amino acid within an xcex1-helical domain.
The present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes eEF-2 kinase; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the eEF-2 kinase has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 5 (SEQ ID NO: 1, 3, and 9).
The human and murine DNA sequences of the eEF-2 kinase gene of the present invention or portions thereof, may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species. The present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for the eEF-2 kinase gene. For example, the probes may be prepared with a variety of known vectors, such as the phage xcex vector. The present invention also includes the preparation of plasmids including such vectors, and the use of the DNA sequences to construct vectors expressing antisense RNA or ribozymes which would attack the mRNAs of any or all of the DNA sequences set forth in FIG. 5 (SEQ ID NO: 1, 3, and 9). Correspondingly, the preparation of antisense RNA and ribozymes are included herein.
The present invention also includes eEF-2 kinase proteins having the activities noted herein, and that display the amino acid sequences set forth and described above and selected from SEQ ID NO: 2, 4, and 10.
In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding eEF-2 kinase, and more particularly, the complete DNA sequence determined from the sequences set forth above and in SEQ ID NO: 1, 3, and 9.
According to other preferred features of certain preferred embodiments of the present invention, a recombinant expression system is provided to produce biologically active animal or human eEF-2 kinase.
The present invention naturally contemplates several means for preparation of eEF-2 kinase, including as illustrated herein known recombinant techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope. The isolation of the cDNA and amino acid sequences disclosed herein facilitates the production of eEF-2 kinase by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.
The invention includes an assay system for screening of potential drugs effective at attenuating eEF-2 kinase activity of target mammalian cells by interrupting or potentiating the phosphorylation of eEF-2. In one instance, the test drug could be administered to a cellular sample along with ATP carrying a detectable label on its xcex3-phosphate that gets transferred to eEF-2, or a peptide substrate, by eEF-2 kinase. Quantification of the labeled eEF-2 or peptide substrate is diagnostic of the candidate drug""s efficacy. A further embodiment would provide for the assay to be performed using a purely in vitro system comprised of eEF-2 kinase , ATP or labeled ATP, eEF-2 or peptide analog of a portion of eEF-2 or MHC, appropriate buffer, and detection reagents and/or instrumentation to detect and quantify the extent of eEF-2 kinase-directed phosphorylation activity.
The assay system could more importantly be adapted to identify drugs or other entities that are capable of binding to the eEF-2 kinase and/or its cognate phosphorylation target (e.g. eEF-2), either in the cytoplasm or in the nucleus, thereby inhibiting or potentiating eEF-2 kinase activity and its resultant phenotypic outcome. Such an assay would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity. For example, such drugs might be used to treat various carcinomas or other hyperproliferitive pathologies.
The present invention likewise extends to antibodies against specifically phosphorylated eEF-2 kinase targets (e.g. eEF-2 or peptide), including naturally raised and recombinantly prepared antibodies. These antibodies and there labeled counterparts are included within the scope of the present invention for their particular ability in detecting eEF-2 kinase activity via detection of the phosphorylated product by ELISA or any other immunoassay known to the skilled artisan.
In the instance where a radioactive label, such as the isotopes 3H, 14C, 32p, 33P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re are used, known currently available counting procedures may be utilized. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized calorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
In a further embodiment, the present invention contemplates antagonists of the activity of eEF-2 kinase. In particular, an agent or molecule that inhibits phosphorylation of eEF-2. In a specific embodiment, the antagonist can be a peptide comprising sequences, or sequence variants adjacent to, and including, the phosphorylation site in either eEF-2 or MHC. It is anticipated that these peptides would be competitive inhibitors of eEF-2 kinase""s cognate target. In still a further embodiment, the invention contemplates antisense drugs such that sequences complementary to the eEF-2 kinase mRNA inhibit production of functional eEF-2 kinase. In a specific embodiment, the antisense drug may be a complementary oligonucleotide (DNA, RNA, or hybrid thereof), which may or may not be modified so as to have the following characteristics: 1) enhanced hybridization kinetics; 2) tighter binding to complementary sequence than its unmodified counterpart; and/or, 3) resistance to nucleases. In another specific embodiment, the antisense drug may be a complementary oligonucleotide (DNA, RNA, or hybrid thereof), that has the ability to cleave its target sequence either by ribozyme, or ribozyme-like, activity, or by nuclease activity imparted on the antisense drug by physical attachment to anyone of a number of nucleases.
More specifically, the therapeutic method generally referred to herein could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions that may comprise effective inhibitors of eEF-2 kinase activity, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
Accordingly, it is a principal object of the present invention to provide a method and an associated assay system for screening potential inhibitors of eEF-2 kinase activity.
It is a further object of the present invention to provide antibodies to the phosphorylated eEF-2 kinase target, and methods for their preparation, including recombinant means.
It is a further object of the present invention to provide a method for detecting eEF-2 kinase activity in mammals in which invasive, spontaneous, or idiopathic pathological states are suspected to be present.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of eEF-2 kinase, so as to alter the adverse consequences of such presence or activity, or where beneficial, to enhance such activity.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of eEF-2 kinase, so as to treat or avert the adverse consequences of invasive, spontaneous or idiopathic pathological states.
It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon a sequence complementary to that of the eEF-2 kinase mRNA, which would form the basis for an antisense therapeutic that can reduce expression, and thus activity, of eEF-2 kinase.
It is yet another object of the invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon peptide analogs of eEF-2 phosphorylation target amino acid sequences. It is anticipated that certain peptide analogs may act as efficacious competitive inhibitors of eEF-2 phosphorylation.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.